Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in lysosomal compartment.

<p>U937 cells were treated with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h, with lysosomal marker (Lyso Tracker Green DND-26, 50 nM) applied for the last 30 min of incubation. Cells were treated with labeled oxLDL and oxLDL-IC at 18 µg/ml and 24 µg/ml, respectively. Live cells were washed with DPBS then suspended in sealed capillaries and visualized using confocal microscopy. Arrows point at co-localization of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC with lysosomal compartment.</p

Lipid moiety of oxLDL-IC but not oxLDL co-localizes with induced HSP70/70B' in endosomal vesicles.

<p>RAW 264.7 cells were transfected with HSP70-GFP (<b>A</b>) or HSP70B'-GFP (<b>B</b>), then treated with DiI-oxLDL (24 µg/ml), DiI-oxLDL-IC (32 µg/ml), or DPBS vehicle in serum-free DMEM for 3 h. Alexa 633-transferrin (10 µg/ml) was then added for an additional 2 h. Cells were then fixed with 4% formaldehyde, washed with DPBS, and visualized using confocal microscopy.</p

Differential effect of oxLDL and oxLDL-IC on mitochondrial membrane potential and ROS/RNS production.

<p>U937 cells were grown in phenol red-free IMDM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml streptomycin, and IFN-γ (200 ng/ml) for 18 h then treated with oxLDL (90 µg/ml), oxLDL-IC, KLH-IC (120 µg/ml) in IMDM up to 5 h. (<b>A</b>) Detection of H₂O₂: Cells were treated with CM-H2DCFDA (5 µM), and Mito Tracker (100 nM) for 30 and 15 min, respectively, prior to conclusion of incubation time with the treatments (5 h). Cells were fixed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. (<b>B</b>) Detection of NO: IFN-γ-treated cells were incubated with L-arginine (100 µM), and DAF-FM diacetate (10 µM) for 1 h, then washed with IMDM twice. Cells were then treated with oxLDL, oxLDL-IC, and KLH-IC as indicated above. Mito Tracker (100 nM) were added 15 min prior to conclusion of incubation time with the treatments (90 min). Live cells were washed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope.</p

Characterization of oxLDL labeling and uptake by U937 cells.

<p>(<b>A</b>) Migration of fluorescently labeled oxLDL analyzed by agarose gel electrophoresis: lane 1: native LDL, lane 2: oxLDL, lane 3: DiI-oxLDL, lane 4: DiO-oxLDL, lane 5: Alexa 546-oxLDL, lane 6: LPDS. (<b>B</b>) Uptake of fluorescently labeled oxLDL. U937 cells were treated with labeled oxLDL: DiO-oxLDL, DiI-oxLDL, or Alexa 546-oxLDL (24 µg/ml) for 5 h then fixed with 4% formaldehyde and visualized in sealed capillaries using confocal microscopy. (<b>C</b>) FACS analysis showing dose-dependent uptake of labeled oxLDL 90 min post treatment in U937 cells. Each data point represents the mean ± range of duplicate determinations (1×10⁴ cells/determination), and data presented are representative of four independent experiments.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in endosomal vesicles.

<p>U937 cells were treated with endosomal marker (Alexa 488-transferrin, green) and with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h. Cells were treated with Alexa 488-transferrin (5 µg/ml) and with either labeled oxLDL (24 µg/ml) or labeled oxLDL-IC (32 µg/ml), fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 laser scanning confocal microscope. Arrows point at co-localization of lipid and apolipoprotein moities of oxLDL and oxLDL-IC with Alexa 488-transferrin.</p

Lipid moieties of oxLDL and oxLDL-IC co-localize when administered sequentially but not simultaneously.

<p>U937 cells were incubated with DiI-oxLDL-IC (red) and DiO-oxLDL (green) (<b>A</b>) sequentially and (<b>B</b>) in parallel in U937 cells. They were incubated for a total of 5 h. Sequential experiment involved 2 h incubation of oxLDL-IC (32 µg/ml), prior to addition of oxLDL (24 µg/ml) for 3 h. Lyso Tacker Blue DND-22, (50 nM) was used and applied for the last 30 min of incubation. Cells were fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. Arrows point at co-localization of oxLDL in the lysosomal compartment in the upper panel, and the co-localization of oxLDL and oxLDL-IC in the lower panel.</p